Anti-proprotein convertase subtilisin kexin type 9 (anti-PCSK9) compounds and methods of using the same in the treatment and/or prevention of cardiovascular diseases

ABSTRACT

Disclosed are compounds that modulate the physiological action of the proprotein convertase subtilisin kexin type 9 (PCSK9), as well as therapeutic methods for use of such compounds to reduce LDL-cholesterol levels and/or for the treatment and/or prevention of cardiovascular disease (CVD), including treatment of hypercholesterolemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a § 371 application of PCT/US2017/038069,filed Jun. 19, 2017, which in turn claims benefit of U.S. ProvisionalPatent Application No. 62/352,701, filed Jun. 21, 2016. The entiredisclosure of each of the foregoing applications is incorporated byreference herein.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made with support from the National Heart,Lung and Blood Institute (NHLBI) under SBIR Grant No. HL096167. The U.S.Government has certain rights in this invention.

FIELD OF INVENTION

The present invention relates to compounds that modulate thephysiological action of the proprotein convertase subtilisin kexin type9 (PCSK9), including its interaction with the low density lipoproteinreceptor (LDLR). More specifically, the invention relates tocompositions comprising small molecule modulators of PCSK9 function andmethods of using these modulators as a medicament. The small moleculemodulators of PCSK9 function can be used therapeutically to lowerLDL-cholesterol levels in blood, and can be used in the preventionand/or treatment of cholesterol and lipoprotein metabolism disorders,including familial hypercholesterolemia, atherogenic dyslipidemia,atherosclerosis, and, more generally, cardiovascular disease (CVD).

BACKGROUND OF INVENTION

Cardiovascular diseases are the leading cause of death, withatherosclerosis being the leading cause of cardiovascular diseases.Atherosclerosis is a disease of the arteries and is responsible forcoronary heart disease associated with many deaths in industrializedcountries. Several risk factors for coronary heart disease have now beenidentified: dyslipidemia, hypertension, diabetes, smoking, poor diet,inactivity and stress. Dyslipidemia is elevation of plasma cholesterol(hypercholesterolemia) and/or triglycerides (TGs) or a low high-densitylipoprotein (HDL) level that contributes to the development ofatherosclerosis. It is a metabolic disorder that is proven to contributeto cardiovascular disease. In the blood, cholesterol is transported inlipoprotein particles, where the low-density lipoprotein (LDL)cholesterol (LDL-C) is considered “bad” cholesterol, whileHDL-cholesterol (HDL-C) is known as “good” cholesterol. Lipid andlipoprotein abnormalities are extremely common in the general populationand are regarded as a highly modifiable risk factor for cardiovasculardisease, due to the influence of cholesterol on atherosclerosis. Thereis a long-felt significant unmet need with respect to CVD with 60-70% ofcardiovascular events, heart attacks and strokes occurring despite thetreatment with statins (the current standard of care inatherosclerosis). Moreover, new guidelines suggest that even lower LDLlevels should be achieved in order to protect high-risk patients frompremature CVD (1).

The establishment of a link between PCSK9 and cholesterol metabolism wasrapidly followed by the discovery that selected mutations in the PCSK9gene caused autosomal dominant hypercholesterolemia (2), suggesting thatthe mutations confer a gain-of-function (3) by increasing the normalactivity of PCSK9. This was supported by the experiment in which wildtype and mutant PCSK9 (S 127R and F216L) were expressed at high levelsin the livers of mice; hepatic LDLR protein levels fell dramatically inmice receiving either the wild type or mutant PCSK9 (4, 5). Noassociated reductions in LDLR mRNA levels were observed, indicating thatoverexpression of PCSK9, whether mutant or wild type reduces LDLRsthrough a post-transcriptional mechanism.

Given that gain-of-function mutations in PCSK9 causehypercholesterolemia, it was reasonable to ask if loss-of-functionmutations would have the opposite effect and result inhypocholesterolemia. Three loss-of-function mutations in PCSK9 (Y142X,L253F, and C679X) were identified in African-Americans (6). Thesemutations reduce LDL-C levels by 28% and were shown to decrease thefrequency of CHD (defined as myocardial infarction, coronary death orcoronary revascularization) by 88%. Rashid et al. (7) studied themechanism of loss-of-function mutations in mice where PCSK9 wasinactivated. They reported that these knockout mice showed increasedhepatic LDLR protein (but not mRNA), increased clearance of circulatinglipoproteins and reduced plasma cholesterol levels. Structure-functionrelationship analysis of the naturally occurring mutations in PCSK9 hasalso provided insights into the mechanism of action of PCSK9.Interestingly, mutations in PCSK9 that were found to be associated withthe greatest reductions in LDL-C plasma levels are those that preventthe secretion of mature PCSK9 by disrupting its synthesis (Y142X),autocatalytic processing (L253F), or folding (C679X) (8). The Y142Xmutation produces no detectable protein because it occurs early in thetranscript and is predicted to initiate nonsense-mediated mRNA decay.

Mutations in the catalytic domain (L253F) interfere with theautocatalytic cleavage of the protein. In cells expressing thePCSK9-253F, the amount of mature protein was reduced compared to that incells expressing PCSK9-WT, suggesting that the mutation inhibitsautocatalytic cleavage. The L253F mutation is near the catalytic triad(PCSK9 is a serine protease), therefore it might disrupt the active site(8). Inasmuch as autocatalytic cleavage of PCSK9 is required for exportof the protein out of the ER, the L253F mutation delays transport ofPCSK9 from the ER to the cell surface. The nonsense mutation (C679X) inPCSK9, which truncates the protein by 14 amino acids, did not interferewith protein processing, but the mature protein accumulates in the cellsand none is secreted, suggesting that the protein is cleaved normallybut is misfolded and is retained in the ER (8, 9).

The mechanism by which PCSK9 causes the degradation of the LDLR has notbeen fully elucidated. However, it is clear that the protease activityof PCSK9 is not required for LDLR degradation (10, 11). Li et al. (10)have co-expressed the prodomain and the catalytic domain in trans, andshowed that the secreted PCSK9 was catalytically inactive, yet it isfunctionally equivalent to the wild-type protein in lowering cellularLDL uptake and LDLR levels. Similar studies were also reported by McNuttet al. (11). Furthermore, Zhang et al. (12) has mapped PCSK9 binding tothe EGF-A repeat of the LDLR, and showed that such binding decreases thereceptor recycling and increases its degradation. They also reportedthat binding to EGF-A domain was calcium-dependent and increaseddramatically with reduction in pH from 7 to 5.2. Kwon et al. (13)determined the crystal structure of PCSK9 in complex with theLDLR-EGF-AB (EGF-A and EGF-B). The structure shows a well-defined EGF-Adomain, but the EGF-B domain is disordered and absent from theirelectron density map. The EGF-A domain binds to the PCSK9 catalyticdomain at a site distant from the catalytic site, and makes no contactwith either the C-terminal domain or the prodomain (14).

Several strategies have been proposed for targeting PCSK9 (15). Strategy1: mRNA knockdown approaches include the use of antisenseoligonucleotides or RNAi. Antisense oligonucleotides administered tomice reduced PCSK9 expression by >90% and lowered plasma cholesterollevels by 53% (16). A single intravenous injection of an RNAi deliveredin lipidoid nanoparticles to cynomologous monkeys reduced plasma PCSK9levels by 70% and plasma LDL-C levels by 56% (17). Strategy 2: is toprevent binding of PCSK9 to the LDLR on the cell surface with a smallmolecule, a peptide, or an antibody directed against PCSK9. Adding EGF-Afragments to cultured cells inhibits the ability of exogenously addedPCSK9 to mediate LDLR degradation.

Strategy 3: is to develop small-molecule inhibitors of the PCSK9processing. Despite evidence that the catalytic activity of PCSK9 is notrequired for LDLR degradation (11), an intracellular inhibitor of PCSK9catalytic activity should be effective, since autocatalytic processingof PCSK9 is required for secretion of the protein from the ER. Followingits synthesis, PCSK9 undergoes an autocatalytic cleavage reaction thatclips off the prodomain, but the prodomain remains attached to thecatalytic domain (18, 19). The autocatalytic processing step is requiredfor the secretion of PCSK9 (20), likely because the prodomain serves asa chaperone and facilitates folding. The continued attachment of theprodomain partially blocks the substrate binding pocket of PCSK9 (18,19). McNutt et al. (21) demonstrated that antagonism of secreted PCSK9increases LDLR expression in HepG2 cells. They show that anFH-associated LDLR allele (H306Y) that results in a gain-of-functionmutation is due to an increase in the affinity of PCSK9 to the LDLR,which would lead to enhanced LDLR destruction, and decreased plasmaLDL-C clearance. Furthermore, they were able to show elegantly thatblocking the secreted PCSK9 with LDLR (H306Y) sub-fragment resulted inan increase in the level of LDLR in cultured HepG2 cells. Therefore,PCSK9 acts as a secreted factor to cause LDLR degradation, and a smallmolecule inhibitor that interferes with the autocatalytic process shoulddecrease the amount of mature secreted PCSK9. This invention relates toidentification of small molecules that down-regulate the function ofPCSK9 using Strategy 2.

Currently (22-24), there are FDA approved injectable PCSK9 monoclonalantibody antagonists on the market. These are Regeneron/Sanofi'sPRALUENT (alirocumab) and Amgen's REPATHA (evolocumab), both of whichare fully human anti-PCSK9 monoclonal antibodies.

Pfizer's bococizumab, which is being developed collaboratively withHalozyme, is furthest ahead at Phase III. Eli Lily's humanized mAbstarted Phase II trials. These monoclonal antibodies approaches followStrategy 2 using injectable antibodies instead of small molecules.

Strategy 2 is also being pursued by several other companies (25).

SUMMARY OF THE INVENTION

This invention relates to therapeutic applications of small moleculesthat selectively interact with and down modulate PCSK9 function. In afirst embodiment, the agents used in the practice of this invention havethe general formula I:A-(CONR_(a))—B  (I)

wherein A is selected from the group consisting of:

wherein Ra is independently selected from the group consisting of H andlower alkyl; R₁ is independently selected from the group consisting ofH, halogen, nitro, cyano, hydroxyl, and optionally substituted amino,alkoxy, alkoxycarbonyl, alkoxyalkyl, alkylthio, alkylthioalkyl, acyl,carboxy, amido, aminocarbonyl, monoalkylaminocarbonyl,dialkylaminocarbonyl, monoalkylaminosulfinyl, dialkylaminosulfinyl,monoalkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino,hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl, and, optionallysubstituted, lower alkyl, cycloalkylalkyl, aralkyl, heterocyclylalkyl,heteroarylalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heterocycle,heteroaryl, or taken together with an adjacent R₁ forms an optionallysubstituted 5-7 membered carbocycle, aryl, heterocycle, or heteroaryl.R₂ is selected from the group consisting of H and optionally substitutedlower alkyl; with the proviso that A is not a 2-substituted indole.

B is selected from the group consisting of optionally substitutedcycloalkyl, aryl, heterocycle, and heteroaryl and the pharmaceuticallyacceptable salts and all stereoisomers of the compound.

In one embodiment, the invention provides a method for the treatment orprophylaxis of hypercholesterolemia and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease in apatient in need of such treatment comprising administering to such apatient a therapeutically effective amount of a compound of formula I,above.

In another embodiment, the method of the invention involvesadministration of at least one compound of the following formula II:

wherein R₁ is independently selected from the group consisting of H, andoptionally substituted (C₁-C₃)-alkyl, (C₁-C₃)-alkoxyalkyl, aryloxyalkyl,(C₁-C₃)-alkylthioalkyl, arylthioalkyl, aryl, and heteroaryl; R₃ isindependently selected from the group consisting of H, halogen, nitro,cyano, (C₁-C₃)-alkoxy, (C₁-C₃)-alkoxycarbonyl, (C₁-C₃)-alkoxyalkyl,aryloxyalkyl, (C₁-C₃)-alkylthio, (C₁-C₃)-alkylthioalkyl, arylthioalkyl,(C₁-C₃)-acyl, carboxy, amido, aminocarbonyl, monoalkylaminocarbonyl,dialkylaminocarbonyl, monoalkylaminosulfonyl, dialkylaminosulfonyl,alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy,alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl, and, optionallysubstituted, lower alkyl, cycloalkylalkyl, aralkyl, heterocyclylalkyl,heteroarylalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heterocycle,heteroaryl or taken together with and adjacent R₃ forms an optionallysubstituted 5-7 membered carbocycle, aryl, heterocycle, or heteroaryl;R₄ is independently selected from the group consisting of H, halogen,(C₁-C₃) alkyl and (C₁-C₃) alkoxy; R₅ is independently selected from thegroup consisting of H and CONR₆R₇, wherein R₆ and R₇ are independentlyselected from the group consisting of H and optionally substitutedalkyl, cycloalkylalkyl, aralkyl, heterocyclylalkyl, heteroarylalkyl,cycloalkyl, alkenyl, alkynyl, aryl, heterocycle, and heteroaryl, or i)taken together forms an optionally substituted 5-7 membered heterocyclecontaining one or more heteroatoms; or ii) taken together with anadjacent R₄ forms an optionally substituted 5-7 membered heterocycle.

In a more preferred embodiment, the method of the invention involvesadministration of at least one compound of the following formula III:

wherein R₁ is independently selected from the group consisting of H orCH₃; R₃ is independently selected from the group consisting of H, or(C₁-C₃)-alkoxy; R₆ and R₇ are independently selected from the groupconsisting of H, CH₃, and optionally substituted aryl, wherein aryl isselected from the group consisting of

wherein R₈ is selected from the group consisting of CO₂H, CONHR₉,2-oxazole, 2-oxazoline, 2-benzoxazole; R₉ is H or CH₃.

In yet another embodiment, the method of the invention involvesadministration of at least one compound of the following formula IV:

wherein R₁ is H or optionally substituted (C₁-C₃)-alkyl; R₃ isindependently selected from the group consisting of H, halogen, oroptionally substituted (C₁-C₃)-alkoxy; R₄ is H; and R₉ is (C₁-C₃)-alkyl.

In a further embodiment, the present invention provides a compound ofthe formula V:

including pharmaceutically acceptable salts and stereoisomers of saidcompound, wherein X_(a) and X_(b) are independently selected from thegroup of H or CH₃;

Y_(a), Y_(b) and Y, may be the same or different and are selected fromthe group of H, halogen or (C₁-C₃)-alkoxy;

Za, Zb and Zc are the same or different and are selected from the groupof H, halogen, (C₁-C₃)-alkoxy, (C₁-C₄)-alkyl, COOH, CONR₁₀R₁₁,2-oxazole, 2-oxazoline and 2-benzoxazole; R₁₀ and R₁₁ are independentlyselected from the group of H, optionally substituted (C₁-C₃)-alkyl andoptionally substituted aryl; n=1 or 2, and when n=1, CONR₁₀R₁₁ may forma N-substituted succinimide ring fused to the benzene ring to which saidCONR₁₀R₁₁ is attached, wherein the succinimide substituent is optionallysubstituted (C₁-C₃) alky or aryl, with the proviso that said formuladoes not include

DESCRIPTION OF DRAWINGS AND TABLES

FIG. 1 sets forth the structure of selected compounds of the formula I,namely 2-carboxanilide pyrroles and 2-carboxanilide indoles, that havean effect on LDLR upregulation as compared to control while having noeffect on PCSK9 processing and secretion, and show in vitro inhibitionof PCSK9/LDLR interaction at 100 μM>20%. Structures for SBC-115,210,SBC-115,211, SBC-115,228, SBC-115,232, SBC-115,229, SBC-115,230,SBC-115,235, SBC-115,240, SBC-115,242, SBC-115,243, SBC-115,249,SBC-115,293, SBC-115,307, and SBC-115, 332 are provided.

FIG. 2 sets forth the structure of preferred compounds of the formulaII-IV, namely 2-carboxanilide benzofurans, that have an effect on LDLRupregulation as compared to control while having no effect on PCSK9processing and secretion, and show in vitro inhibition of the PCSK9/LDLRinteraction (IC₅₀, μM) >10 μM but 50 μM. Structures for SBC-115,203,SBC-115,251, SBC-115,256, SBC-115,419, SBC-115,422, SBC-115,425,SBC-115,427, SBC-115,429, SBC-115,430, SBC-115,435, SBC-115,437,SBC-115,438, SBC-115,439, SBC-115,440, SBC-115,441, SBC-115,442,SBC-115,444, and SBC-115,446 are provided.

FIG. 3 sets forth the structure of the most preferred compounds of theformula II-IV that have an effect on LDLR upregulation as compared tocontrol while having no effect on PCSK9 processing and secretion, andshow in vitro inhibition of the PCSK9/LDLR interaction (IC₅₀, μM)<10 μM.Structures for SBC-115,202, SBC-115,270, SBC-115,271, SBC-115,337,SBC-115,341, SBC-115,415, SBC-115,417, SBC-115,418, SBC-115,421,SBC-115,423, SBC-115,424, SBC-115,426, SBC-115,431, SBC-115,432,SBC-115,433, SBC-115,436, SBC-115,443, SBC-115,445, and SBC-115,447 areprovided.

FIG. 4 shows the effect of different compounds on the PCSK9/LDLRinteraction: An in vitro ELISA assay kit was utilized (Circulex). Forscreening inhibitors of the PCSK9/LDLR interaction, differentconcentrations (0.01 nM-100 μM) of selected compounds were incubatedwith His-tagged PCSK9 and then added to wells that were pre-coated withrecombinant LDLR-AB domain. After incubation, the plate was washed andthe amount of recombinant His-tagged PCSK9 was measured using thebiotinylated anti-His-tag and horseradish peroxidase conjugatedStreptavidin, and quantitated using a BioTek Synergy 2 plate reader. Theeffect of each compound on the PCSK9 binding to the recombinant LDLR-ABdomain was calculated.

FIG. 5 shows the effect of different compounds on PCSK9 synthesis,processing and secretion in HEK293 transfected cells. HEK-293T cellswere seeded into 96 well plates in a DMEM containing 10% Fetal BovineSerum media and incubated overnight at 37° C. Cells were transientlytransfected with cDNA construct using the Lipofectamine-LTX. Compounds(25 μM) or vehicle were added, followed by additional 43 hours ofincubation. Cellular PCSK9, secreted PCSK9, and cell viability wereanalyzed as described in Example 2 below.

FIG. 6 shows increased degradation of the LDLR by PCSK9. HEK-293T cellswere seeded in a DMEM containing 10% Fetal Bovine Serum media andincubated overnight at 37° C. Cells were transiently transfected withMock (lanes 1 and 2), PCSK9 (lanes 3 and 4), LDLR & PCSK9 (lanes 5 and6), and LDLR (lanes 7 and 8) cDNA constructs using theLipofectamine-LTX. Cells were incubated for an additional 72 hrs, andcells and media were analyzed as in text.

FIG. 7 shows upregulation of LDLR by PCSK9 antagonists. HEK-293T cellswere seeded in a DMEM containing 10% Fetal Bovine Serum media andincubated overnight at 37° C. Cells were transiently transfected withLDLR & PCSK9 cDNA constructs using the Lipofectamine-LTX as describedabove. After 24 hrs, cells were treated with different compounds andincubated for an additional 48 hrs. Cells were assayed as describedabove for LDLR expression.

FIG. 8 shows effect of different compounds on LDLR upregulation in HepG2cells. HepG2 cells were seeded into 96 well plates in a MEM containing10% Fetal Bovine Serum media and incubated overnight at 37° C. Cellswere transiently transfected with PCSK9 cDNA constructs using theLipofectamine-LTX. Compounds were added, followed by additional 43 hoursof incubation. The cells were lysed and analyzed for LDLR expression andcell viability determined as described above.

FIG. 9 shows increased uptake of Fluorescent Dil-LDL using fourinhibitors in HepG2 cells. The SBC compounds were validated for theirability to increase uptake of Fluorescent Dil-LDL in HepG2 cells. Thedata show an increase in the Fluorescent Dil-LDL uptake using 1.2 μM ofthe compound.

FIG. 10 shows the effect of SBC-115,337 on LDL cholesterol levels of 8mice fed high fat diet (HFD) with and without LPS compared to animalsfed regular diet. C57/Black 6 mice and BalB/C mice were maintained onthe HFD for 4 weeks. Blood plasma was collected at day 1 prior toinjection of the SBC compounds. Blood plasma was collected at day 4, at4 hours after the LPS injection, and again at 24 hours after LPSinjection. Plasma cholesterol, LDL-C, HDL-C, and triglyceride levelswere measured enzymatically.

FIG. 11 shows the SAR around SBC-115,337 on the PCSK9/LDLR interaction:an in vitro ELISA assay kit was utilized as described in FIG. 4.

FIG. 12 shows increased uptake of Fluorescent Dil-LDL using variousinhibitors in HepG2 cells. The SBC compounds were validated for theirability to increase uptake of Fluorescent Dil-LDL in HepG2 cells asdescribed in FIG. 9.

FIG. 13 shows the effect of different compounds on PCSK9 synthesis,processing and secretion in HEK293/PCSK9 transfected cells. The SBCcompounds were validated for their ability to affect PCSK9 synthesis,processing and secretion as described in relation to FIG. 5.

FIG. 14 shows the upregulation of LDLR by PCSK9 antagonists. HEK-293Tcells were assayed as described in relation to FIG. 7.

FIG. 15A-FIG. 15B shows the effect of SBC-115,418 on LDL cholesterollevels in C57/Black6 mice fed high fat diet:C57BL/6 mice were dividedinto 2 groups of 5 animals in each; control group received PBS;SBC-group received 10 mg/kg oral daily for 5 days. Blood plasma wascollected at day 1 prior to injection of the compounds. Blood plasma wascollected again at day 5. Plasma LDL-C levels was measuredenzymatically. (FIG. 15A) Representing the actual change in the LDL-C inmg/dL and (FIG. 15B) representing the % reduction of LDL-C after theadministration of SBC-115,418 for 5 days.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to small molecules that down regulate thefunction of extracellular proprotein convertase subtilisin kexin type 9(PCSK9), including its interaction with the low density lipoprotein(LDL) receptor (LDLR), and methods of using these antagonists as amedicament. The small molecule modulators of PCSK9 function can be usedtherapeutically to lower LDL-cholesterol levels in blood, and can beused in the prevention and/or treatment of cholesterol and lipoproteinmetabolism disorders, including familial hypercholesterolemia,atherogenic dyslipidemia, atherosclerosis, and, more generally,cardiovascular disease (CVD).

As used herein, the term “alkyl” is a branched or unbranched saturatedhydrocarbon chain moiety. “Lower alkyl” denotes branched or unbranchedhydrocarbon chains, having 1 to about 8 carbons, such as, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,2-methylpentyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethyl pentyl,octyl, 2,2,4-trimethylpentyl and the like. “Substituted alkyl” includesan alkyl group which may be substituted with one or more substituentgroups which are attached commonly to such chains, such as, hydroxy,halogen, mercapto or thio, cyano, alkylthio, carboxy, carbalkoxy, amino,nitro, alkoxy, or optionally substituted, alkenyl, alkynyl,heterocyclyl, aryl, heteroaryl, and the like to form alkyl groups suchas trifluoro methyl, 3-hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl,carboxymethyl, cyanobutyl, phenethyl, benzyl and the like.

The term “halogen” or “halo” as used herein alone or as part of anothergroup refers to chlorine, bromine, fluorine, and iodine.

The term “alkoxy” refers to alkyl-O—, in which alkyl is as definedabove.

The term “alkylthio” refers to alkyl-S—, in which alkyl is as definedabove.

The terms “amino”, “monoalkylamino”, “dialkylamino” refer to the moiety—NR′R″, in which R′ and R″, each independently represents H, alkyl oraryl, all as defined herein.

The term “carboxy” refers to the moiety —C(═O)OH.

The term “carbalkoxy” refers to the moiety —C(═O)O-alkyl, in which alkylis as defined above.

The term “amino (monoalkylamino-, dialkylamino-) carbonylamino” refersto the moiety —NHC(═O)NR′R″, in which R′R″, each independentlyrepresents H, alkyl or aryl, all as defined herein.

The term “carbamato” refers to the moiety —NR′C(═O)OR″, in which R′ andR″, each independently represents H, alkyl or aryl, all as definedherein.

The term “amino (monoalkylamino, dialkylamino) carbonyl” (also“carboxamido”) refers to the moiety —C(═O)NR′R″, in which R′ and R″ eachindependently represents H, alkyl, or aryl, all as defined herein.

The term “amido” refers to the moiety NRC(═O)—R″, in which R″ and R″,each independently represents H, alkyl or aryl, all as defined herein.

The term “alkylsulfonyl” refers to the moiety —S(═O)2-alkyl, in whichalkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)2-alkyl, whereinalkyl is as previously defined.

The term “amino (monoalkylamino-, dialkylamino-) sulfinyl” refers to themoiety S(═O)NR′R″ in which R′ and R″ each independently represents H,alkyl or aryl, all as defined herein.

The term “amino (monoalkylamino-, dialkylamino-) sulfonyl” refers to themoiety S(═O)2NR′R″, in which R′ and R″ each independently represents H,alkyl or aryl, all as defined herein.

The term “alkylsulfonylamino” refers to the moiety —NHS(═O)2-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyloxy” refers to the moiety —OS(═O)2OH.

The term “alkoxysulfonyloxy” refers to the moiety —OS(═O)2O-alkyl, inwhich alkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)2-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyl” refers to the moiety —S(═O)2OH.

The term “alkoxysulfonyl” refers to the moiety —S(═O)2O-alkyl, whereinalkyl is as previously defined.

The term “alkylsulfonylalkyl” refers to the moiety -alkyl-S(═O)2-alkyl,wherein alkyl (each instance) is as previously defined.

The term “amino (monoalkylamino-, dialkylamino-) sulfonylalkyl” refersto the moiety alkyl-S(═O)2-NR′R″, wherein alkyl is as previouslydefined, and R″ and R″ each independently represents H, alkyl or aryl,all as defined herein.

The term “amino (monoalkylamino-, dialkylamino-) sulfinylalkyl” refer tothe moiety alkyl-S(═O)—NR′R″, wherein alkyl is as previously defined,and R′ and R″ each independently represents H, alkyl or aryl, all asdefined herein.

Unless otherwise indicated, the term “cycloalkyl” as employed hereinalone or as part of another group includes saturated or partiallyunsaturated (containing 1 or more double bonds) cyclic hydrocarbongroups (carbocyclic) containing 1 to 3 rings, including monocyclicalkyl,bicyclicalkyl and tricyclicalkyl, containing a total of 3 to 20 carbonsforming the rings, preferably 3 to 10 carbons, forming the ring andwhich may be fused to 1 or 2 aromatic rings as described for aryl, whichinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl cyclododecyl and cyclohexenyl.

“Substituted cycloalkyl” includes a cycloalkyl group which may besubstituted with 1 or more substituents such as halogen, alkyl,substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl, aryloxy,cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino,amino, nitro, cyano, thiol and/or alkylthio and/or any of thesubstituents included in the definition of “substituted alkyl.”

Unless otherwise indicated, the term “alkenyl” as used herein by itselfor as part of another group refers to straight or branched chain of 2 to20 carbons, preferably 2 to 12 carbons, and more preferably 2 to 8carbons in the normal chain, which include one or more double bonds inthe normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-butenyl,4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl,4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl,4,8,12-tetradecatrienyl, and the like. “Substituted alkenyl” includes analkenyl group which may be substituted with one or more substituents,such as the substituents included above in the definition of“substituted alkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “alkynyl” as used herein by itselfor as part of another group refers to straight or branched chain of 2 to20 carbons, preferably 2 to 12 carbons and more preferably 2 to 8carbons in the normal chain, which include one or more triple bonds inthe normal chain, such as 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl,3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl,3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl, 4-dodecynyl and the like.“Substituted alkynyl” includes an alkynyl group which may be substitutedwith one or more substituents, such as the substituents included abovein the definition of “substituted alkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “aryl” or “Ar” as employed hereinalone or as part of another group refers to monocyclic and polycyclicaromatic groups containing 6 to 10 carbons in the ring portion (such asphenyl or naphthyl including 1-naphthyl and 2-naphthyl) and mayoptionally include one to three additional rings fused to a carbocyclicring, such as a cycloalkyl ring or fused to an aryl or heterocyclic ringor substituted forms thereof.

“Substituted aryl” includes an aryl group which may be substituted withone or more substituent groups, such as halo, alkyl, haloalkyl (e.g.,trifluoromethyl), alkoxy, haloalkoxy (e.g., difluoromethoxy), alkenyl,alkynyl, cycloalkyl-alkyl, heterocyclo-alkyl, aryl, heteroaryl,arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy, alkoxycarbonyl,alkylcarbonyl, arylcarbonyl, arylalkenyl, aminocarbonyl,monoalkylaminocarbonyl, dialkylaminocarbonyl, aminocarbonylaryl,arylthio, arylsulfinyl, arylazo, heteroarylalkyl, heteroarylalkenyl,heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro, cyano, amino,substituted amino wherein the amino includes 1 or 2 substituents (whichare optionally substituted alkyl, aryl or any of the other substituentsmentioned in the definitions), thiol, alkylthio, heteroarylthio,arylthioalkyl, alkoxyarylthio, alkylaminocarbonyl, arylaminocarbonyl,alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino,arylcarbonylamino, arylsulfinylalkyl, arylsulfonylamino orarylsulfonaminocarbonyl and/or any of the alkyl substituents referred toabove.

Unless otherwise indicated, the term “heteroaryl” or “Het” as usedherein alone or as part of another group refers to a 5- or 7-memberedaromatic ring which includes 1, 2, 3 or 4 hetero atoms such as nitrogen,oxygen or sulfur and such rings fused to an aryl, cycloalkyl, heteroarylor heterocycloalkyl ring and includes possible N-oxides. Examples ofheteroaryl groups include pyrrolyl, furanyl, thienyl, pyrazolyl,imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,isooxazolyl, thiazolyl, isothiazolyl, thiadiazolyl and oxadiazolyl.Examples of fused heteroaryl groups include quinoline, isoquinoline,indole, isoindole, carbazole, acridine, benzimidazole, benzofuran,benzoxazole, isobenzofuran, benzothiophene, phenanthroline, purine, andthe like. “Substituted heteroaryl” includes a heteroaryl group which maybe substituted with 1 to 4 substituents, such as the substituentsincluded above in the definition of “substituted alkyl” “substitutedcycloalkyl” and “substituted aryl”.

The term “heterocyclo”, “heterocycle” or “heterocyclic ring,” as usedherein alone or as part of another group, represents an unsubstituted orsubstituted stable 5- to 7-membered monocyclic ring system which may besaturated or partially unsaturated, and which consists of carbon atomsand from one to four heteroatoms selected from N, O or S, and whereinthe nitrogen and sulfur heteroatoms may optionally be oxidized, and thenitrogen heteroatom may optionally be quaternized. “Substitutedheterocyclo (or heterocycle or heterocyclic ring) includes aheterocyclic group which may be substituted with 1 to 4 substituents,such as the substituents included above in the definition of“substituted alkyl” “substituted cycloalkyl” and “substituted aryl”. Theheterocyclic ring may be attached at any heteroatom or carbon atom whichresults in the creation of a stable structure. Examples of suchheterocyclic groups include, but are not limited to, piperidinyl,piperazinyl, oxopiperazinyl, oxopiperidinyl, oxopyrrolidinyl,oxoazepinyl, azepinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide andthiamorpholinyl sulfone.

The term “optionally substituted” is used herein to signify that achemical moiety referred to, e.g., alkyl, aryl, heteroaryl, may beunsubstituted or substituted with one or more groups including, withoutlimitation, lower alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl, aryl,haloaryl, heterocycle, heterocycloalkyl, heteroaryl, hydroxyl, amino,monoalkylamino, dialkylamino, alkoxy, halogen, haloalkoxy, aryloxy,aryloxyalkyl, alkylaryloxy, arylalkoxy, alkoxyaryl, carboxy, carbalkoxy,carboxamido, aminocarbonyl, monoalkylaminocarbonyl,dialkylaminocarbonyl, monoalkylaminosulfinyl, dialkylaminosulfinyl,monoalkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino,hydroxysulfonyloxy, alkoxysulfonyloxy, alkylsulfonyloxy,hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl and the like. Thechemical moieties of formulas I-V, above, that may be optionallysubstituted include lower alkyl, alkenyl, alkynyl, cycloalkyl,arylalkyl, aryl, heterocycle, and heteroaryl. For example, optionallysubstituted alkyl would comprise both propyl and 2-chloro-propyl.Additionally, “optionally substituted” is also inclusive of embodimentswhere the named substituent or substituents have multiple substituentsrather than simply a single substituent. For example, optionallysubstituted aryl would comprise both phenyl and3-bromo-4-chloro-6-ethyl-phenyl.

Unless expressly indicated otherwise, all references herein to alkyl andaryl groups also include the substituted forms thereof.

Among the substituents of the A moiety represented by R1 in Formula I,above, preferred are H or optionally substituted (C1-C3)-alkyl at the3-position, and where the 4- and 5-positions are joined together to forman optionally substituted 6-membered aromatic ring; and particularlypreferred substituents are H or CH₃ at the 3-position, and a fusedbenzene ring at the 4- and 5-positions optionally substituted withhalogen and (C₁-C₃)-alkoxy.

Among the substituents represented by R3 in Formulas II, III and IV,above preferred are H, halogen, or (C₁-C₃)-alkoxy; and particularlypreferred are H and (C1-C3) alkoxy.

As used herein, the term “subject” includes both humans and animals. Asused herein, the term “PCSK9” refers to any form of the protein PCSK9,including PCSK9 mutants and variants, which retain at least part ofPCSK9 activity or function. Unless otherwise indicated, such as byspecific reference to human PCSK9, PCSK9 refers to all mammalian speciesof native sequence PCSK9, e.g., human, porcine, bovine, equine, canineand feline. One exemplary human PCSK9 sequence is found as UniprotAccession Number Q8NBP7 (SEQ ID NO:16).

As used herein, a “modulator of PCSK9 function” refers to a smallmolecule that is able to inhibit PCSK9 biological activity or function,and/or downstream pathway(s) mediated by PCSK9 signaling, includingPCSK9-mediated down-regulation of the LDLR, and PCSK9-mediatedinhibition of the decrease in LDL blood clearance. A modulator of PCSK9function encompasses compounds that block, antagonize, suppress orreduce (to any degree including significantly) PCSK9 biologicalactivity, including downstream pathways mediated by PCSK9 signaling,such as LDLR interaction and/or elicitation of a cellular response toPCSK9. For purpose of the present invention, it will be explicitlyunderstood that the term “modulator of PCSK9 function” encompasses allthe previously identified terms, titles, and functional states andcharacteristics whereby the PCSK9 itself, a PCSK9 biological activity(including but not limited to its ability to mediate any aspect ofinteraction with the LDLR, down regulation of LDLR, and inhibit thedecrease in blood LDL clearance), or the consequences of the biologicalactivity, are substantially nullified, decreased, or neutralized in anymeasurable degree. In some embodiments, a modulator of PCSK9 functionbinds PCSK9 and prevents its interaction with the LDLR or its secretion.In other embodiments, a modulator of PCSK9 function binds to the activesite of PCSK9 to stabilize its zymogen and prevent autoprocessing. Infurther embodiments, a modulator of PCSK9 function decreases or blocksPCSK9 mediated down-regulation of the LDLR; inhibits the PCSK9-mediateddecrease in LDL blood clearance; increases LDL clearance in media bycultured hepatocytes; increases blood LDL clearance by the liver invivo; improves patients' sensitivity to other LDL lowering drugs,including statins; is synergistic to other LDL lowering drugs, includingstatins; and blocks PCSK9 interaction with other yet to be identifiedfactors. Examples of modulators of PCSK9 function are provided herein.

The compounds used in the method of the invention can be administered assalts, which are also within the scope of this invention.Pharmaceutically acceptable (i.e., non-toxic, physiologicallycompatible) salts are preferred. If the compounds of the method of thepresent invention have, for example, at least one basic center, they canform acid addition salts. These are formed, for example, with stronginorganic acids, such as mineral acids, for example sulfuric acid,phosphoric acid or a hydrohalic acid, with strong organic carboxylicacids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which areunsubstituted or substituted, for example, by halogen, for exampleacetic acid, such as saturated or unsaturated dicarboxylic acids, forexample oxalic, malonic, succinic, maleic, fumaric, phthalic orterephthalic acid, such as hydroxycarboxylic acids, for exampleascorbic, glycolic, lactic, malic, tartaric or citric acid, such asamino acids, for example aspartic or glutamic acid or lysine orarginine, or benzoic acid, or with organic sulfonic acids, such as(C1-C4) alkyl or arylsulfonic acids which are unsubstituted orsubstituted, for example by halogen, for example methyl- orpara-toluene-sulfonic acid. Corresponding acid addition salts can alsobe formed having plural basic centers, if desired. The compounds used inthe method of the present invention having at least one acid group (forexample COOH) can also form salts with suitable bases. Representativeexamples of such salts include metal salts, such as alkali metal oralkaline earth metal salts, for example sodium, potassium or magnesiumsalts, or salts with ammonia or an organic amine, such as morpholine,thiomorpholine, piperidine, pyrrolidine, a mono, di or tri-loweralkylamine, for example ethyl, tert-butyl, diethyl, diisopropyl,triethyl, tributyl or dimethyl-propylamine, or a mono, di or trihydroxylower alkylamine, for example mono, di or triethanolamine.

Corresponding internal salts may also be formed.

Preferred salts of the compounds described herein which contain a basicgroup include monohydrochloride, hydrogensulfate, methanesulfonate,phosphate or nitrate.

Preferred salts of the compounds described herein which contain an acidgroup include sodium, potassium and magnesium salts and pharmaceuticallyacceptable organic amines.

All stereoisomers of the compounds which may be used in the methodsdescribed herein, either in a mixture or in pure or substantially pureform, are considered to be within the scope of this invention. Thecompounds of the present invention can have asymmetric centers at any ofthe carbon atoms including any one of the R substituents. Consequently,compounds used in the method of the invention can exist in enantiomericor diastereomeric forms or in mixtures thereof. The processes forpreparation of such compounds can utilize racemates, enantiomers ordiastereomers as starting materials. When diastereomeric or enantiomericproducts are prepared, they can be separated by conventional methods forexample, chromatographic, chiral HPLC or fractional crystallization.

As used herein, the term “pharmacophore” refers to the ensemble ofsteric and electronic features that are necessary to ensure the optimalsupramolecular interactions with a specific biological target structureand to trigger, activate, block, inhibit or modulate the biologicaltarget's biological activity, as the case may be. See, IUPAC, Pure andApplied Chemistry (1998) 70: 1129-1143.

As used herein, the term “pharmacophore model” refers to arepresentation of points in a defined coordinate system wherein a pointcorresponds to a position or other characteristic of an atom or chemicalmoiety in a bound conformation of a ligand and/or an interactingpolypeptide, protein, or ordered water molecule. An ordered watermolecule is an observable water in a model derived from structuraldetermination of a polypeptide or protein. A pharmacophore model caninclude, for example, atoms of a bound conformation of a ligand, orportion thereof. A pharmacophore model can include both the boundconformations of a ligand, or portion thereof, and one or more atomsthat interact with the ligand and are from a bound polypeptide orprotein.

Thus, in addition to geometric characteristics of a bound conformationof a ligand, a pharmacophore model can indicate other characteristicsincluding, for example, charge or hydrophobicity of an atom or chemicalmoiety. A pharmacophore model can incorporate internal interactionswithin the bound conformation of a ligand or interactions between abound conformation of a ligand and a polypeptide, protein, or otherreceptor including, for example, van der Waals interactions, hydrogenbonds, ionic bonds, and hydrophobic interactions. A pharmacophore modelcan be derived from two or more bound conformations of a ligand.

As used herein, the term “ligand” refers to any compound, composition ormolecule that interacts with the ligand binding domain of a receptor andmodulates its activity. A “ligand” may also include compounds thatmodulate the receptor without binding directly to it.

In carrying out the method of the invention, the above-describedcompounds may be administered as such, or in a form from which theactive agent can be derived, such as a prodrug.

A prodrug is a derivative of a compound described herein, thepharmacologic action of which results from the conversion by chemical ormetabolic processes in vivo to the active compound.

The term “prodrug esters” as employed herein includes esters andcarbonates formed by reacting one or more hydroxyls of compounds used inthe method of the invention with alkyl, alkoxy, or aryl substitutedacylating agents employing procedures known to those skilled in the artto generate acetates, pivalates, methylcarbonates, benzoates and thelike. Any compound that can be converted in vivo to provide thebioactive agent (i.e., a compound of formula I) is a prodrug within thescope and spirit of the invention. Various forms of prodrugs are wellknown in the art.

A comprehensive description of prodrugs and prodrug derivatives aredescribed in: (a) The Practice of Medicinal Chemistry, Camille G.Wermuth et al., Ch 31 (Academic Press, 1996); (b) Design of Prodrugs,edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Designand Development, P. Krogsgaard-Larson and H. Bundgaard, eds., Ch. 5,pgs, 113-191 (Harwood Academic Publishers, 1991).

The therapeutic agent used in practicing the method of the invention isadministered in an amount sufficient to induce the desired therapeuticeffect in the recipient thereof. Thus the term “therapeuticallyeffective amount” as used herein refers to an amount of a therapeuticagent which is sufficient to treat or prevent a condition treatable byadministration of one or more of the compounds of formulas I—V or aprodrug thereof. Preferably, the therapeutically effective amount refersto the amount appropriate to treat a PCSK9-associated condition, i.e. tobring almost a detectable therapeutic or preventative or ameliorativeeffect. The effect may include, for example, treatment or prevention ofthe conditions described herein.

The compound(s) described herein may be administered at a dose in rangefrom about 0.01 mg to about 200 mg/kg of body weight per day. A dose offrom 0.1 to 100, and preferably from 1 to 30 mg/kg per day in one ormore applications per day should be effective to produce the desiredresult. By way of example, a suitable dose for oral administration wouldbe in the range of 1-30 mg/kg of body weight per day, whereas a typicaldose for intravenous administration would be in the range of 1-10 mg/kgof body weight per day. Of course, as those skilled in the art willappreciate, the dosage actually administered will depend upon thecondition being treated, the age, health and weight of the recipient,the type of concurrent treatment, if any, and the frequency oftreatment. Moreover, the effective dosage amount may be determined byone skilled in the art on the basis of routine empirical activitytesting to measure the bioactivity of the compound(s) in a bioassay, andthus establish the appropriate dosage to be administered.

The compounds used in the method of the invention will typically beadministered from 1-4 times a day, so as to deliver the above-mentioneddaily dosage. However, the exact regimen for administration of thecompounds described herein will necessarily be dependent on the needs ofthe individual subject being treated, the type of treatment administeredand the judgment of the attending medical specialist.

In one aspect, the invention provides a method for treating orpreventing hypercholesterolemia, and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease, in anindividual comprising administering to the individual an effectiveamount of a modulator of PCSK9 function that antagonizes circulatingPCSK9.

In a further aspect, the invention provides an effective amount of amodulator of PCSK9 function that antagonizes circulating PCSK9 for usein treating or preventing hypercholesterolemia, and/or at least onesymptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease,in an individual. The invention further provides the use of an effectiveamount of a modulator of PCSK9 function that antagonizes extracellularor circulating PCSK9 in the manufacture of a medicament for treating orpreventing hypercholesterolemia, and/or at least one symptom ofdyslipidemia, atherosclerosis, CVD or coronary heart disease, in anindividual.

The methods of the invention use a modulator of PCSK9 function, whichrefers to any molecule that blocks, suppresses or reduces (includingsignificantly reduces) PCSK9 biological activity, including downstreampathways mediated by PCSK9 signaling, such as elicitation of a cellularresponse to PCSK9.

A modulator of PCSK9 function should exhibit any one or more of thefollowing characteristics: (a) bind to PCSK9; (b) decrease or blockPCSK9 interaction with the LDLR; (c) decrease or block secretion ofPCSK9; (d) decrease or block PCSK9 mediated down-regulation of the LDLR;(e) inhibit the PCSK9-mediated decrease in LDL blood clearance, (f)increase LDL clearance in media by cultured hepatocytes, (g) increaseblood LDL clearance by the liver in vivo, (h) improve patients'sensitivity to other LDL lowering drugs, including statins, (i) issynergistic to other LDL lowering drugs, including statins; and (j)block PCSK9 interaction with other yet to be identified factors.

In general, the compound(s) used in the method of the invention can beadministered to achieve modulation of PCSK9 function by using anyacceptable route known in the art, either alone or in combination withone or more other therapeutic agents. Thus, the active agent(s) can beadministered orally, buccally, parenterally, such as by intravenous orintra-arterial infusion, intramuscular, intraperitoneal, intrathecal orsubcutaneous injection, by liposome-mediated delivery, rectally,vaginally, by inhalation or insufflation, transdermally or by oticdelivery.

The orally administered dosage unit may be in the form of tablets,caplets, dragees, pills, semisolids, soft or hard gelatin capsules,aqueous or oily solutions, emulsions, suspensions or syrups. Suitabledosage forms for parenteral administration include injectable solutionsor suspensions, suppositories, powder formulations, such asmicrocrystals or aerosol spray. The active agent may also beincorporated into a conventional transdermal delivery system.

As used herein, the expression “physiologically compatible carriermedium” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface agent agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants, fillers and the like as suited for the particular dosageform desired. Remington: The Science and Practice of Pharmacy, 20^(th)edition (A. R. Genaro et al., Part 5, Pharmaceutical Manufacturing, pp.669-1015 (Lippincott Williams & Wilkins, Baltimore, Md./Philadelphia,Pa.) (2000)) discloses various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Except insofar as any conventional pharmaceutical carriermedium is incompatible with the PCSK9 modulators used in the presentinvention, such as by producing an undesirable biological effect orotherwise interacting in an deleterious manner with any othercomponent(s) of a formulation comprising such compounds, its use iscontemplated to be within the scope of this invention.

For the production of solid dosage forms, including hard and softcapsules, the therapeutic agent may be mixed with pharmaceuticallyinert, inorganic or organic excipients, such as lactose, sucrose,glucose, gelatine, malt, silica gel, starch or derivatives thereof,talc, stearic acid or its salts, dried skim milk, vegetable, petroleum,animal or synthetic oils, wax, fat, polyols, and the like. For theproduction of liquid solutions, emulsions or suspensions or syrups onemay use excipients such as water, alcohols, aqueous saline, aqueousdextrose, polyols, glycerine, lipids, phospholipids, cyclodextrins,vegetable, petroleum, animal or synthetic oils. For suppositories onemay use excipients, such as vegetable, petroleum, animal or syntheticoils, wax, fat and polyols. For aerosol formulations, one may usecompressed gases suitable for this purpose, such as oxygen, nitrogen andcarbon dioxide. The pharmaceutical composition or formulation may alsocontain one or more additives including, without limitation,preservatives, stabilizers, e.g., UV stabilizers, emulsifiers,sweeteners, salts to adjust the osmotic pressure, buffers, coatingmaterials and antioxidants.

The present invention further includes controlled-release,sustained-release, or extended-release therapeutic dosage forms foradministration of the active agent, which involves incorporation of theactive agent into a suitable delivery system. This dosage form controlsrelease of the active agent(s) in such a manner that an effectiveconcentration of the active agent(s) in the bloodstream may bemaintained over an extended period of time, with the concentration inthe blood remaining relatively constant, to improve therapeutic resultsand/or minimize side effects. Additionally, a controlled-release systemwould provide minimum peak to trough fluctuations in blood plasma levelsof the active agent.

In pharmaceutical compositions used in practicing the method of theinvention, the active agent(s) may be present in an amount of at least0.5 and generally not more than 95% by weight, based on the total weightof the composition, including carrier medium and/or supplemental activeagent(s), if any. Preferably, the proportion of active agent(s) variesbetween 30-90% by weight of the composition.

Compounds for use in practicing this invention include those of formulasI-IV, above. More preferred are the compounds set out in FIG. 2. Mostpreferred are the compounds set out in FIG. 3.

Some of the compounds described herein are obtainable from commercialsources, such as Life Chemicals (SBC-115,202), Enamine (SBC-115,270 andSBC-115,271), ChemBridge (SBC-115,337), and Princeton Biomolecular(SBC-115,341). Other preferred compounds and almost all of the mostpreferred compounds were not available from commercial sources but weresynthesized using methods known to those skilled in the art of organicsynthesis.

The methods of the present invention will normally include medicalfollow-up to determine the therapeutic or prophylactic effect broughtabout in the subject undergoing treatment with the compound(s) and/orcomposition(s) described herein.

The activities of compounds described herein have been experimentallydemonstrated. The following examples are provided to describe theinvention in further detail. These examples are provided forillustrative purposes only and are not intended to limit the inventionin any way.

Example 1 Test for LDLR/PCSK9 Binding

Testing of compounds acquired from commercial sources was performed inour binding assay for their potential to inhibit the PCSK9/LDLRinteraction. Initial screening was done using 100M of the compounds.Each compound was added to the binding reaction mixture in triplicateand assayed as described above. From the screening, a number ofcompounds were identified to have an effect on LDLR upregulation ascompared to control (see Example 3 vide infra) while having no effect onPCSK9 processing and secretion (see Example 2 vide infra). Exemplary2-carboxyanilide indoles and 2-carboxanilide pyrroles of the formula Ithat had >20% inhibition at 100 μM are shown in FIG. 1. Fifty five (55)structurally related 2-carboxyanlide benzofurans (compounds of theformulas II-IV) were also acquired. Compounds that inhibited thePCSK9/LDLR interaction were selected and analyzed using differentcompound concentrations ranging from 100 μM to 0.01 nM. The most potentbenzofuran compounds were SBC-115,270, SBC-115,271, SBC-115,337 andSBC-115,202 with IC50's of 7, 7.3, 0.5 and 9.5 PM, resp. (FIG. 4). Basedon this preliminary SAR, an additional 34 compounds were designed andsynthesized around SBC-115,337, and the compounds were tested in ourbinding assay as described in FIG. 4. From the screening, a number ofbenzofuran compounds were determined to have IC50's>10 μM but less</=50μM (FIG. 2). Still more potent compounds had IC50's<10 μM (FIG. 3). FIG.11 and Table 1 shows the IC50's of the best ten of these compounds.Compounds that exhibited low or submicromolar potency were selected forfurther evaluation in multiple different cell based assays.

TABLE 1 Summary of the IC50 of compounds around SBC-115,337 CompoundIC-50 (μM) SBC-115,337 0.6 SBC-115,415 5.9 SBC-115,418 1.6 SBC-115,4238.2 SBC-115,424 1.7 SBC-115,432 1.1 SBC-115,433 2.4 SBC-115,440 18.8SBC-115,441 19.9 SBC-115,443 5.5 SBC-115,444 14.5 SBC-115,445 2.2

Example 2 Test for Secreted PCSK9

The increase in the level of recombinant LDLR in the presence of PCSK9by the above compounds could be either due to inhibiting the binding ofPCSK9 to the LDLR or by inhibiting the processing and secretion ofPCSK9. To eliminate the possibility these compounds interfere with PCSK9synthesis, processing or secretion, we tested the effect of thesecompounds on the processing and secretion of PCSK9 as described above.HEK-293T cells were seeded into 96-well plates in a DMEM containing 10%Fetal Bovine Serum media and incubated overnight at 37° C. Cells weretransiently transfected with cDNA construct using the Lipofectamine-LTX.Compounds (25 μM) or vehicle were added, followed by additional 43 hoursof incubation. Cellular PCSK9, secreted PCSK9, and cell viability wereanalyzed for PCSK9 secretion using western blot analysis, imaged andquantitated using a LAS-4000 (GE). Results from four selected compoundsare shown in FIG. 5. All of these compounds exhibited no effect on thesynthesis, processing and secretion of PCSK9 either in the cells or intothe media (FIG. 5).

Example 3 Test for LDLR Upregulation

We used our own recombinant assay which demonstrates that co-expressionof PCSK9 and LDLR DNA in HEK-293 cells results in a decrease in theexpression level of intracellular LDLRs. We have constructed theexpression vector of human LDLR under the control of the cytomegaloviruspromoter-enhancer (pCMV-LDLR). In addition, a construct containing thePCSK9 (pCMV-PCSK9-FLAG) was described above. These constructs were usedto transiently transfect mammalian cells, and both cell lysate andsupernatant were subjected to SDS-PAGE and immunoblot analysis using ananti-PCSK9 or LDLR antibody. The data from the blot showed that cellsthat were transfected with only pCMV-PCSK9-FLAG expressed both theunprocessed (cells) and processed (media) PCSK9 (FIG. 6). Cells thatwere transfected with only pCMV-LDLR showed expression of the LDLR inthe cells (FIG. 6). However, cells that were transfected with bothpCMV-PCSK9-FLAG and pCMV-LDLR showed disappearance of the intracellularLDLR band (FIG. 6), which provides further evidence that the presence ofPCSK9 results in degradation of LDLR or chaperones it to the degradationpathway. Addition of inhibitors of PCSK9 processing to the latter cellsshould result in decreased degradation of the LDLR and the appearance ofthe 160K Dalton band on the gel. Using this assay, we tested ourcompounds for their ability to reduce the degradation of the LDLR.HEK-293 cells were used in this assay. They were grown in 96-well platesovernight, and transfected with LDLR/PCSK9.

Compounds dissolved in DMSO or vehicle were added to the culture media,and incubated for 24-48 hours; cells were lysed. Cell lysates weresubjected to quantitation using the above immunoassay. FIG. 7 shows theeffect of four compounds on the intracellular recombinant LDLRupregulation. Compounds SBC-115,270, SBC-115,271, SBC-115,337 andSBC-115,202 exhibited a 5 to 10 fold up-regulation at 1.2 μM.

Testing confirmed that these compounds are capable of upregulating theendogenously expressed LDLR in HepG2 cells. HepG2 cells transfected withPCSK9 were cultured in 96-well plates at a density of 30,000 cells perwell. The next day, cells are treated with selected screening compoundsor vehicle. Cells were incubated for 48 hrs and then subjected toquantitation using an LDL receptor-polyclonal antibody and analyzed asdescribed above. The data in FIG. 8 shows that these compounds exhibitedan increase in the level of LDLR as compared to cells treated with thesame volume of DMSO with a 4-8 fold upregulation of LDLR.

Example 4 Uptake of Dil-LDL in HepG2 Cells In Situ

In order to confirm that these PCSK9 antagonists upregulate functionalLDLR, we tested the ability of these compounds to enhance the uptake offluorescent Dil-LDL in HepG2 cells. Briefly, HepG2 cells transfectedwith PCSK9 (FIG. 4) were plated and allowed to grow overnight. Compoundswere added to the cells followed by the addition of fluorescent Dil-LDL.Cells were washed extensively, and the fluorescent Dil-LDL taken up bythe cells were measured using the Synergy 2 plate reader (FIG. 9 andFIG. 12). As shown in FIG. 9, our compounds (SBC-115,337, SBC-115,270,SBC-115,271 and SBC-115,202) exhibited an increase in fluorescentlylabeled LDL uptake in HepG2 treated cells at 1.2 μM. Further analysis ofadditional selected compounds from our binding studies (Table 1) showedthat four compounds (SBC-115,418, SBC-115,424, SBC-115,432 andSBC-115,433) exhibited significant increases in the Dil-LDL uptake at1.2 μM compound concentration (FIG. 12). These compounds were selectedfor further analysis in the in vivo studies.

Example 5 Test for LDLR Upregulation

We used our recombinant LDLR upregulation assay (Example 3) to validatethat SBC-115,418, SBC-115,424, SBC-115,432 and SBC-115,433 exhibited asignificant increase in the level of LDLR as compared to cells treatedwith the same volume of DMSO (FIG. 13).

Example 6 Test for Secreted PCSK9

The increase in the level of recombinant LDLR of the four selectedcompounds (SBC-115,418, SBC-115,424, SBC-115,432 and SBC-115,433) shownin FIG. 14 is not due to its effect on synthesis and secretion of PCSK9,but rather on its effect on the LDLR/PCSK9 interaction (see example 2).

Example 7 Test for Cell Viability

All compounds that upregulate the endogenously expressed LDLR were usedto test for in situ cell viability. HEK-293T cells or HepG2 cells wereseeded in 96-well plates in a cell media containing 10% Fetal BovineSerum and incubated overnight at 37° C. Compounds (25 μM) were added tocells after 24 hours and incubated for an additional 48 hours. Cellviability was assayed using Resazurin (Sigma 199303) and a Synergy-IIplate reader. Compounds that showed cell toxicity were excluded.

Example 8 Test for Efficacy in an Animal Model

SBC-115,337 was tested for efficacy in male mice (C57BL/6 mice). Micewere housed as four animals per cage under climate-controlled conditionsof temperature (20-24° C.), humidity (60-70%), and alternating 12 hlight/dark cycles. The mice were divided into five groups as shown inFIG. 10. One group was fed commercial chow diet (Prolab RMH 3000, PMIfeeds, St. Louis, Mo.) to serve as a negative control, while the otherfour groups were fed high fat diet (TD.06414), which provides 60% ofcalories from fat. Water was provided ad libitum. Plasma was collectedonce weekly to monitor the level of LDL. After 4 weeks of feeding a highfat-diet, mice were randomly assigned to one of several groups such thatthe average LDL levels were equal among different groups. One of thefour groups of mice fed high fat diet was treated with vehicle andserved as a positive control, whereas the second group was treated dailywith 3 mg/kg of SBC-115,337 subcutaneously for 4 days. The third groupof mice fed high fat diet was treated with vehicle and LPS (5 mg/Kg) andserved as a LPS positive control, whereas the fourth group was treateddaily with 3 mg/kg SBC-115,337+LPS (5 mg/Kg) subcutaneously for 4 days.Blood samples (75 al) were collected 4 days after drug administrationfrom the retro-orbital venous plexus via heparinized capillary tubescontaining 2 USP units of ammonium heparin per tube (Carolina,Burlington, N.C.). Plasma was separated immediately by centrifugation(5,000×g) for 5 min at room temperature and then kept at −80° C. untilassayed for lipid profile. Plasma cholesterol, LDL-C, HDL-C, andtriglyceride levels were measured enzymatically.

Our data demonstrated that SBC-115,337 lowered LDL-C levels in mice thatare fed high fat diet and treated or untreated with LPS. FIG. 10 showsdata obtained with SBC-115,337 indicating a 20-25% reduction in LDL-Clevels after 4 days relative to high fat diet animal levels.

Similarly, SBC-115,418 was tested in mice as described above. C57BL/6mice were divided into 4 groups of 5 animals in each; two base linegroups, a control group that received PBS and a SBC-group that received10 mg/kg oral daily for 5 days. Blood plasma was collected at day 1prior to injection of the compounds. Blood plasma was collected again atday 5. Plasma LDL-C levels were measured enzymatically. (A) Representingthe actual change in the LDL-C in mg/dL and (B) representing the %reduction of LDL-C after the administration of SBC-115,418 for 5 days.Our data demonstrated that SBC-115,418 lowered LDL-C levels in mice thatare fed high fat diet. FIG. 15A and FIG. 15B shows data obtained withSBC-115,418 indicating a 20-25% reduction in LDL-C levels after 5 daysof daily oral administration of 10 mg/kg of SBC-115,418 relative to highfat diet animal levels.

The foregoing specification includes citations to certain publications,which are provided to indicate the state of the art to which thisinvention pertains. The entire disclosure of each of the citedpublications is incorporated by reference herein.

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing disclosure. The presentinvention is, therefore, not limited to the particular embodimentsdescribed and/or exemplified, but is capable of considerable variationand modification without departure from the scope of the appendedclaims. Furthermore, the transitional terms “comprising”, “consistingessentially of” and “consisting of”, when used in the appended claims,in original and amended form, define the claim scope with respect towhat unrecited additional claim elements or steps, if any, are excludedfrom the scope of the claim(s). The term “comprising” is intended to beinclusive or open-ended and does not exclude any additional, unrecitedelement, method, step or material. The term “consisting of” excludes anyelement, step or material other than those specified in the claim and,in the latter instance, impurities ordinarily associated with thespecified material(s). The term “consisting essentially of” limits thescope of a claim to the specified elements, steps or material(s) andthose that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. All compositions and methodsof use thereof that embody the present invention can, in alternateembodiments, be more specifically defined by any of the transitionalterms “comprising,” “consisting essentially of,” and “consisting of.”

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What is claimed is:
 1. A method for treating hypercholesterolemia,and/or at least one symptom of dyslipidemia, atherosclerosis, CVD orcoronary heart disease in a patient in need of said treatment, themethod comprising administering to said patient a therapeuticallyeffective amount of a compound of the formula:

wherein R₁ is selected from the group consisting of H, and optionallysubstituted (C₁-C₃)-alkyl, (C₁-C₃)-alkoxyalkyl, aryloxyalkyl,(C₁-C₃)-alkylthioalkyl, arylthioalkyl, aryl, and heteroaryl; each R₃ isindependently selected from the group consisting of H, halogen,(C₁-C₃)-alkyl, (C₁-C₃)-alkoxy, and optionally substituted aryl, or R₃taken together with an adjacent R₃ forms an optionally substituted 5-7membered aryl ring; each R₄ is independently selected from the groupconsisting of H, halogen, (C₁-C₃) alkyl and (C₁-C₃) alkoxy; R₅ isCONR₆R₇, wherein R₆ and R₇ are independently selected from the groupconsisting of H and optionally substituted aryl; or a pharmaceuticallyacceptable salt or stereoisomer of the compound.
 2. The method of claim1, comprising administering a therapeutically effective amount of acompound of the formula:

wherein R₁ is selected from the group consisting of H and CH₃; R₃ isindependently selected from the group consisting of H and(C₁-C₃)-alkoxy; and one of R₆ and R₇ is H and the other is

wherein R₈ is selected from the group consisting of CO₂H, CONHR₉,2-oxazole, 2-oxazoline, and 2-benzoxazole; R₉ is H or optionallysubstituted (C₁-C₃)-alkyl; or a pharmaceutically acceptable salt orstereoisomer of the compound.
 3. A method for treatinghypercholesterolemia, and/or at least one symptom of dyslipidemia,atherosclerosis, CVD or coronary heart disease in a patient in need ofsaid treatment, the method comprising administering to said patient atherapeutically effective amount of a compound selected from the groupconsisting ofN-(4-{[4-(1,3-benzoxazol-2-yl)phenyl](methyl)carbamoyl}phenyl)-1-benzofuran-2-carboxamide(SBC-115,423);N-(4-{[4-(1,3-benzoxazol-2-yl)phenyl]carbamoyl}phenyl)-1-benzofuran-2-carboxamide(SBC-115,337);N-(4-{[4-(1,3-benzoxazol-2-yl)phenyl]carbamoyl}phenyl)-5-methoxy-3-methyl-1-benzofuran-2-carboxamide(SBC-115,418);N-{4-[(4-carbamoylphenyl)carbamoyl]phenyl}-1-benzofuran-2-carboxamide(SBC-115,424);N-(4-{[4-(4,5-dihydro-1,3-oxazol-2-yl)phenyl]carbamoyl}phenyl)-1-benzofuran-2-carboxamide(SBC-115,432);N-(4-{[4-(1,3-oxazol-2-yl)phenyl]carbamoyl}phenyl)-1-benzofuran-2-carboxamide(SBC-115,433);7-methoxy-3-methyl-N-(4-{[4-(1,3-oxazol-2-yl)phenyl]carbamoyl}phenyl)-1-benzofuran-2-carboxamide(SBC-115,445); and pharmaceutically acceptable salts or stereoisomersthereof.
 4. The method of claim 3, wherein said compound isN-(4-{[4-(1,3-benzoxazol-2-yl)phenyl]carbamoyl}phenyl)-5-methoxy-3-methyl-1-benzofuran-2-carboxamide(SBC-115,418) or a pharmaceutically acceptable salt or stereoisomerthereof.
 5. The method of claim 3, wherein said compound isN-(4-{[4-(1,3-benzoxazol-2-yl)phenyl](methyl)carbamoyl}phenyl)-1-benzofuran-2-carboxamide(SBC-115,423) or a pharmaceutically acceptable salt or stereoisomerthereof.